Immunomodulating effects of water-soluble extracts of traditional French Alps cheeses on a human T-lymphocyte cell line
Introduction
Since the pioneering work of Metchnikoff (1908), consumption of fermented milk is believed to be useful in maintaining good health. This encouraged many investigators to study the potential role of fermented milk product (such as yogurt) consumption in the prevention of various diseases such as cancer, infection and gastrointestinal disorders (Hosono, Otani, Yasui, & Watanuki, 2002). Because the immune system is an important contributor to all these diseases, an immunostimulatory effect of yogurt has been proposed and investigated in vivo by using mainly animal models and, occasionally, human subjects (Meydani & Ha, 2000). In addition, in vitro cell culture test systems have been employed to screen the direct effect of fermented milk on various cell types of the human immune system. Several in vitro indices of immune function have been determined for different constituents of fermented milk (lactic acid bacteria, water-soluble fraction, etc.). This has allowed for identification of several components, which exert an immunomodulatory effect in vivo: fermented milk bacterial (cell wall polysaccharides, peptidoglycan, teichoic acid) and non-bacterial components (peptides and free fatty acids generated by fermentation, whey protein, calcium) (Ganjam, Thornton, Marshall, & MacDonald, 1997). These studies support the hypothesis that consumption of fermented milk may enhance the immune response, but further work is needed to confirm it and to elucidate the mechanisms involved.
In addition to fermented milk, cheeses constitute another family of milk-derived fermented products. Consumption of cheese is also believed to exert a stimulatory effect on immune system functions, but studies dealing with the immunomodulatory effect of cheese and its components are scarce. A Lactobacillus plantarum strain isolated from ewes’ milk cheese induced in vitro production of several cytokines by human macrophages (Haza, Zabala, & Morales, 2004). In addition to the bacterial cell components, the immunomodulatory effect of cheese could also result from non-bacterial components such as peptides: the peptides present in a lyophilized extract of Gouda cheese suppressed proliferation of cultured human peripheral blood lymphocytes in vitro and induced apoptosis of human promyelocytic leukaemia cells (IL-60) (Meisel & Günther, 1998). The presence of β-casomorphin 1–7 in Brie cheese has been reported by Jarmolowska, Kostyra, Krawczuk, and Kostyra (1999). This opioid peptide derived from β-casein (f60–66) could also exert an effect on the immune system, since it is a ligand of μ-type opioid receptors, which are present on the surface of T-lymphocytes (Brantl, Bläsig, & Henschen, 1981; Chang, Su, Brent, & Chang, 1985).
Several other fragments of bovine casein have been shown to exert an immunomodulatory effect in vitro (Coste et al., 1992; Hata, Higashiyama, & Otani, 1998; Kayser & Meisel, 1996; Meisel & Günther, 1998; Migliore-Samour, Floc’h, & Jollès, 1989; Sandré et al., 2001; Xiao, Jin, & Zhao, 2000) or in vivo (Lahov & Regelson, 1996; LeBlanc, Matar, Valdéz, LeBlanc, & Perdigon, 2002). These fragments could be released by proteolysis during cheese ripening. In the present study, the presence of immunomodulatory compounds was investigated in the water-soluble extracts (WSEs) of four pressed-curd cheeses (Emmental de Savoie, Beaufort, Abondance and Tomme de Savoie). The high casein content of these traditional pressed-curd cheeses from the Alps region (duly described by Berard, Froc, Hyman, and Marchenay (1995)) coupled with their long ripening periods allows for the accumulation of casein fragments in their water-soluble fraction. We chose these four cheeses to evaluate the impact of different cheesemaking parameters, such as the cooking temperature and the ripening period, on the presence of immunomodulatory components. Also of concern are the microflora involved (e.g. moulds and mesophilic bacteria for Tomme de Savoie, thermophilic bacteria for the Swiss-type cheeses Beaufort and Emmental de Savoie). The immunomodulation activity of these four cheeses was assessed by following the proliferation and the metabolic activity of T-lymphocytes cultured in vitro in the presence of cheese WSEs.
Section snippets
Cell line and culture conditions
The frozen human T-lymphocytes (JURKAT E6-1) secreting IL-2 were obtained from the European Collection of Cell Culture (ECACC, Salisbury, UK). The medium consisted of RPMI (Roswell Park Memorial Institute) 1640 (Sigma, St. Louis, MO, USA) supplemented with 5% or 2.5% of foetal calf serum (FCS), glutamine (2mm) and antibiotics (100 U mL−1 penicillin, 100 μg mL−1 streptomycin) (Gibco®, Invitrogen, Cergy-Pontoise, France).
Culture operating conditions
T-lymphocytes were cultured in the above medium at 37 °C in a 95% relative
Results
Human T-lymphocyte proliferation and metabolic activity were evaluated with BrdU and MTT assays, respectively: these two in vitro assays optimized for the cell line were performed at the different growth phases to study the impact of cheese WSEs on the immunomodulation of cells.
Discussion
When examining the effect of cheese WSEs on the proliferation of non-adapted and adapted cells, the following conclusions can be drawn: (i) growth phases of cells stimulated by a cheese WSE were identical for Beaufort and Emmental WSEs at the stationary phase and to a lesser extent the exponential phase, while Abondance and Tomme de Savoie WSEs stimulated the lag phase, (ii) the effects (i.e. the SI value variations) were always more pronounced when the BrdU assays were performed with adapted
Conclusion
If most of the studies evaluated the cell proliferation with a single technique (direct cell count, incorporation of tritiated thymidine, BrdU or MTT assays), a methodology developed in a preliminary study (Durrieu et al., 2005), allowing the assessment of immunomodulation by studying two parameters implicated in the cell growth (DNA synthesis and metabolic activity) simultaneously, was used in the present work. From the four screened cheeses and with the sampling methodology considered within
Acknowledgments
This research was supported by La Région Rhône-Alpes and the Institut Technique Français des Fromages.
References (33)
- et al.
Opioid activities of β-casomorphins
Life Sciences
(1981) - et al.
Isolation of a specific μ-opiate receptor peptide, morphiceptin, from an enzymatic digest of milk proteins
Journal of Biological Chemistry
(1985) - et al.
Identification of C-terminal peptides of bovine β-casein that enhance proliferation of rat lymphocytes
Immunology Letters
(1992) - et al.
Modified colorimetric ninhydrin methods for peptidase assay
Analytical Biochemistry
(1981) - et al.
Biologically active peptides from milk proteins with emphasis on two examples concerning antithrombotic and immunomodulating activities
Journal of Dairy Science
(1993) - et al.
Antiproliferative effects of yogurt fractions obtained by membrane dialysis on cultured mammalian intestinal cells
Journal of Dairy Science
(1997) - et al.
Protective effect and cytokine production of a Lactobacillus plantarum strain isolated from ewes’ milk cheese
International Dairy Journal
(2004) - et al.
Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins
FEBS Letters
(1996) - et al.
Antibacterial and immunostimulating casein-derived substances from milk: Casecidin, isracidin peptides
Food and Chemical Toxicology
(1996) - et al.
Immunomodulating effects of peptidic fractions issued from milk fermented with Lactobacillus helveticus
Journal of Dairy Science
(2002)
Immunologic effects of yogurt
American Journal of Clinical Nutrition
Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays
Journal of Immunological Methods
A peptide derived from bovine β-casein modulates functional properties of bone marrow-derived macrophages from germfree and human flora-associated mice
Journal of Nutrition
Produits du terroir et recettes traditionnelles
Activité protéolytique de souches de lactobacilles thermophiles isolés de levains et de Comté. I. Validation sur minifromages des techniques de laboratoire
Lait
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